Immunostimulatory oligodeoxynucleotides

ABSTRACT

The present invention relates to immunostimulatory oligodeoxynucleotides, vectors and vaccines comprising such oligodeoxynucleotides, to their use as a medicament, to their use in preventing or combating infectious disease, to methods for the detection of such oligodeoxynucleotides and to cells to be used in these methods.

The present invention relates to immunostimulatory oligodeoxynucleotides, vectors and vaccines comprising such oligodeoxynucleotides, to their use as a medicament, to their use in preventing or combating infectious disease, to methods for the detection of such oligodeoxynucleotides and to cells to be used in these methods.

During the past two decades, it has emerged in immunological science that the vertebrate immune system possesses mechanisms to detect microbial infection and to trigger rapid immune activation via the receptor-mediated recognition of unique characteristics of pathogens, the so-called pathogen-associated molecular patterns (PAMPs) interacting with cognate host pathogen recognition receptors (PRRs) (Iwasaki A, Medzhitov R. 2001. Science 327, 291-295. Medzhitov R., 2009. Immunity 30, 766-775).

It is now clear that certain forms of pathogen deoxyribonucleic acid (DNA) are amongst these PAMPs. In 1995 it was reported that non-methylated CpG motifs in bacterial DNA trigger murine B-cell activation (Krieg et al. 1995). This study generated for the first time a link between the specific recognition of bacterial immunostimulatory non-methylated CpG-containing DNA and the previously recognized CpG suppression as well as the widespread CpG methylation in mammalian DNA. The most effective B cell stimulatory non-methylated CpG oligodeoxynucleotide (CpG ODN) was shown to possess the sequence element GACGTT.

The next landmark paper in the field was published by Shizuo Akira's laboratory in Osaka/Japan (Hemmi et al. 2000). By a gene cloning and a targeted gene knockout approach in mice it could be unequivocally shown, that the cellular response in mice to CpG-ODNs is mediated by the toll-like receptor 9 (TLR9). Subsequently it was shown that the CpG-ODNs are agonists for TLR9 signaling predominantly via the NF kappa-B pathway (Medzhitov 2001). In the following decade, quite a number of studies have been published on basic research topics and on general potential immunotherapeutic applications (e. g. reviewed in Krieg 2002, 2003, 2006; Klinman 2004, Vollmer 2005, Wilson et al. 2006, Kindrachuk et al. 2008, Dorn and Kippenberger 2008, Vollmer and Krieg 2009, Wilson et al. 2009). A number of review articles focus on anti-infective applications of CpG-ODNs (Krieg 2007), the use of TLR9 agonists in the treatment of cancer (Krieg 2007, Weiner 2009), TLR9 activation for asthma and allergy treatment (Kline 2007, Kline and Krieg 2008, Fonseca and Kline 2009) and as vaccine adjuvants (Klinman et al. 2004, Klinman 2006, Daubenberger 2007, Wagner 2009, Mutwiri et al. 2009, Klinman et al. 2009).

CpG ODNs have also been described and discussed as immunostimulatory agents and vaccine adjuvants in veterinary applications, particularly in bovines, pigs, sheep, dogs, chicken and fish (Babiuk et al. 2003, Carrington and Secombes 2006, Griebel et al. 2005, Mutwiri et al. 2003, Singh and O'Hagan 2003, Werling and Jungi 2003).

In the field of veterinary uses in chickens, the use of CpG oligodeoxynucleotides in e.g. vaccines to protect chickens against Newcastle Disease has been described (Linghua 2007).

It has recently been shown that in chicken, TLR21 acts as a functional homologue to mammalian TLR9 in the recognition of CpG oligodeoxynucleotides (Brownlie et al., 2009).

The design of specific CpG ODN's as immunomodulators has so far been quite random. This is especially true for non-mammalian CpG ODN's. The reason for this is multi-factorial; first of all there is no knowledge about correlation between immuno modulatory CpG motifs for human TLR's and for TLR's in non-human, let alone non-mammalian species. Secondly, there are no cell-systems available with a sufficiently low background to noise level to selectively test the effects of very low concentrations of CpG ODN's. Moreover, there are no high-throughput screening methods available and even if there were, there is no clear correlation between in vivo versus in vitro efficacy of CpG ODN's as immuno-modulators in non-mammalian species.

Thus, there clearly is a need for novel CpG ODN's that have a high immuno-modulatory effect and therefore are effective in low doses. And there is a need for selective and sensitive CpG ODN selection systems for veterinary purposes that show a correlation between in vitro and in vivo activity of CpG-activity.

It is one of the objectives of the present invention to provide such novel CpG ODN's.

In this respect, one embodiment of the present invention relates to an immunostimulatory non-methylated oligodeoxynucleotide having the general formula ^(5′) [G]_(x) {([T]_(p) T T C G T C [T]_(q)}_(n) [G]_(z) ^(3′) wherein p=1-15, q=1-15, n=2-100, x=3-10 and z=0-10 or a pharmaceutically acceptable salt thereof

An “immunostimulatory non-methylated oligodeoxynucleotide” refers to an oligodeoxynucleotide, which contains a non-methylated cytidine-phosphate-guanosine di-nucleotide sequence that stimulates the initiation of signaling cascades leading to activation of transcription factors such as NF-κB or Interferon Regulatory Factor 3 (IRF3). It is this activation that in turn results in the expression of inflammatory cytokines and other cellular activation events. NF-κB binding sites and gene expression influenced by NF-κB are i.a. described by Schindler and Baichwal (1994).

The term oligodeoxynucleotide means a short nucleic acid polymer of deoxynucleotides; i.e. a molecule comprising a multitude of deoxyriboses, linked to a phosphate group and to an exchangeable organic base. Such an organic base is a substituted pyrimidine or a substituted purine. Examples are cytosine and thymine respectively adenine and guanine.

The oligonucleotides according to the invention may comprise modifications. Examples of such modifications are e.g. modifications in the phosphodiester internucleoside bridge located at the 3′ and/or 5′ end of a nucleoside. Such modifications relate i.a. to the replacement of a phosphodiester by e.g. a phosphorothioate or a phosphorodithioate.

Other modifications are e.g. replacements of a phosphodiester bridge by a dephospho bridge. Examples of dephospho bridges are methylhydroxylamine, formacetal and dimethylenesulfone groups.

Still other modifications are modifications that concern the replacement of a natural nucleoside base by a non-natural nucleoside base such as 5-fluorocytosine, 7-deaza-7-substituted guanine, 7-deaza-8-substituted guanine, 2-thiouracil, dihydrouracil, 5-bromo-cytosine, 6-substituted cytosines, N4-substituted cytosines,

Again other modifications are modifications concerning the replacement of a sugar unit; a β-ribose sugar or a β-D-2′-ribose sugar unit by a modified sugar unit such as e.g. an L-2′-deoxyribose or 2′-L-arabinose.

A text book giving further insight in oligonucleotides is e.g. “PCR Primer: A Laboratory Manual”, Second Edition, 2003, Edited By Carl W. Dieffenbach, National Institute of Allergy and Infectious Diseases; Gabriela S. Dreksler, Uniformed Services University of the Health Sciences, Cold Spring Harbor Laboratory Press ISBN 978-087969654-2.

The structure {([T]_(p) T T C G T C [T]_(q)}_(n) carrying the CpG motif represents the active immunostimulating moiety of an ODN according to the invention. Therefore, the present invention provides immunostimulatory oligodeoxynucleotides that comprise this so-called “backbone”.

It was found that the backbone of an oligodeoxynucleotide according to the invention, the structure {[T]_(p) T T C G T C [T]_(q)}_(n) must be present at least two, preferably three times.

Therefore, n should be at least two. It was also found that the activity of the oligodeoxynucleotides increases when n increases. This effect is leveling when n increases. Basically, the number n of the backbone structure should therefore be at least 2. Preferably, the range of n is 3≦n≦100, merely because of the fact that the longer the synthetic sequence the more difficult it is to make. In practice preferably the range of n is 2≦n≦18. More preferably, the range of n is 3≦n≦18, even more preferably the range of n is 4≦n≦18, still even more preferably the range of n is 5≦n≦18.

The identification of CpG ODN's according to the invention was made possible i.a. by using a more selective detection system than the systems currently in use for the detection of NF-κB activation. Brownlie at al. (2009) describe an NF-κB luciferase based reporter system. Other systems are e.g. based upon IL-8 transcript measurement or cytokine secretion or the detection of NO secretion.

Contrary to this, in the present invention a secreted alkaline phosphatase based detection system (SEAP) was used. SEAP is a reporter enzyme in mammalian systems (Yang et al., 1997). This system turned out to be surprisingly sensitive and in addition surprisingly provides a close correlation between the in vitro and in vivo activities of the CpG ODN's tested. The SEAP system was used with para-nitrophenylphosphate (pNPP) as a substrate.

Another improvement over existing systems was the introduction and stable maintenance in cells of the plasmid carrying the SEAP gene. Up till now, all detection systems used transient transfection of cells with the reporter gene. It is due to the introduction and stable maintenance in cells of the reporter gene that now for the first time a dose/response curve could be made. Such a curve is essential if a reliable comparison between various CpG ODN's activity is to be made.

Therefore, the methods and cell lines described in detail in the Examples section of the present invention allow for the first time to make a reliable side-by-side comparison between various CpG ODN's.

Further details of the system used are given in the Examples section.

Since the present methods and cell lines now allow such reliable side-by-side comparisons between various CpG ODN's, it could be determined that an oligodeoxynucleotide according to the invention wherein p>1 has a higher activity level than when p=1. Therefore, in a preferred form of this embodiment, p>1. A p-value of 2, 3, 4, 5, or 6 are more preferred in that order of preference. Still more preferred are p-values of >6, although the increase in activity levels off. P-values that exceed 15 would be increasingly difficult to synthesize. Therefore, preferably, the value for p should not exceed 15.

It could also be determined that an oligodeoxynucleotide according to the invention wherein q>1 has a higher activity level than when q=1. Therefore, in a preferred form of this embodiment, q>1. A q-value of 2, 3, 4, 5, or 6 are more preferred in that order of preference. Still more preferred are q-values of >6, although the increase in activity levels off.

Q-values that exceed 15 would be increasingly difficult to synthesize. Therefore, preferably, the value for q should not exceed 15.

It was found that an increase in the number of G's ate the 5′-end of the structure ^(5′) [G]_(x) {[T]_(p) T T C G T C [T]_(q) }_(n)[G]_(z) ^(3′) leads to an increase of the activity of the CpG ODN. The value x should be at least 3, but increasing numbers of G's up to 20 G's improve the activity of the CpG ODN. Therefore preferably, x is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 in that increasing order of preference.

It was also found that an increase in the number of G's ate the 3′-end of the structure ^(5′) [G]_(x) {[T]_(p) T T C G T C [T]_(q)}_(n)[G]_(z) ^(3′) leads to a (slight) decrease of the activity of the CpG ODN. The value z should be 10 or less than 10, but decreasing the number of G's down to 0 G's improves the activity of the CpG ODN. Therefore more preferably z is 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0, in that increasing order of preference.

As said above, several kinds of modifications in the phosphodiester internucleoside bridge located at the 3′ and/or 5′ end of a nucleoside are feasible. But basically, depending upon the way of synthesis, usual common types of bonds between two nucleotides are: phosphodiester (PDE) bonds and phosphorothioate (PTO) bonds. In order to improve the stability and the immunostimulatory effect of CpG ODN's, the building blocks of synthetic oligodeoxynucleotides are provided with phosphorothioates, so that they form PTO bonds.

It was surprisingly found, however, that when only the ^(5′) [G]_(x) and the ^(3′) [G]_(z) nucleotides are bound by PTO bonds and the other nucleotides are bound by PDE bonds, the efficacy of the oligodeoxynucleotide according to the invention is further increased. (In such cases, the ^(5′) [G]_(x) to {([T]_(p) T T C G T C [T]_(q)} bond is a PTO bond, while the {[T]_(p) T T C G T C [T]_(q)} to [G]_(x) ^(3′) bond is a PDE bond.)

Therefore, another preferred form of this embodiment relates to oligodeoxynucleotides according to the invention wherein the ^(5′) [G]_(x) and the ^(3′) [G]_(z) nucleotides have a phosphorothioate binding and the other nucleotides have a phosphodiester binding.

It is not necessary that the backbone of oligodeoxynucleotides according to the invention, the structure ^(5′) {[T]_(p) T T C G T C [T]_(q)}_(n) ^(3′) is identical for every n. This means that an oligodeoxynucleotide backbone according to the invention could look i.a. like this: {T T T C G T C T}{T T T C G T C T T}{T T T T C G T C T}. Such a series of three different consecutive different backbones would be indicated as a heteropolymer. A stretch of three identical copies would be called a homopolymer.

Preferably, the oligodeoxynucleotide according to the invention comprises a ^(5′) {[T]_(p) T T C G T C [T]_(q) }_(n) ^(3′) homopolymer.

The CpG oligodeoxynucleotides according to the invention are active in picomolar (sub-nanomolar) amounts; their EC50 is below 1 nM.

The half-maximal effective concentration (EC50) of an oligodeoxynucleotide is the amount of oligodeoxynucleotide that is necessary to induce an amount of the reporter enzyme SEAP (that produces the colored product absorbing at 405 nm) in the reporter cells (HEK293-pNifty2-chickenTLR21 or HD11-pNifTy2Hyg) that gives a half-maximal enzymatic reaction rate. If the EC50 of an oligodeoxynucleotide is below 1 nM in these cells, it is considered to be active in picomolar (sub-nanomolar) amounts.

It is very well possible to link an oligodeoxynucleotide according to the invention to a carrier or hapten, via a reactive chemical group. Such linkage enhances the immunostimulatory effect of the combined molecules.

Mere examples of such components are e.g. digoxigenin, aminohexyl-, Texas red and biotin. Preferred carriers or haptens are 3′- and 5′-labeled Texas red and 5′-labeled digoxigenin. The linkage of oligodeoxynucleotides to haptens/carriers is well-known in the art.

Another embodiment of the invention relates to a vector comprising an immunostimulatory non- methylated oligodeoxynucleotide according to the invention. Such a vector can be a nucleic acid molecule such as a plasmid, a virus, a bacteriophage or any other vector used in molecular biology. Merely as an example: a vector comprising an immunostimulatory non-methylated oligodeoxynucleotide can e.g. be a DNA molecule such as a plasmid that can be multiplied in bacteria, into which an immunostimulatory non-methylated oligodeoxynucleotide according to the invention has been cloned. Such a plasmid preferably has an active origin of replication, causing high numbers of the plasmid to be present in the host. Growing such bacteria on a large scale followed by isolation of the plasmids provides an alternative for the synthetic production of the immunostimulatory non-methylated oligodeoxynucleotide according to the invention.

One of the aims of the present invention is to provide new CpG ODN's that can be used as successful immunostimulating components in vaccines that prevent or combat infectious disease together with an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier.

In general, the term antigen component refers to a composition of matter that comprises at least one epitope that can induce, stimulate or enhance an immune response when administered to a human or an animal.

The antigen component may be any kind of antigen component but preferably is derived from a micro-organism or virus that in its wild-type form is pathogenic to humans or animals.

The antigen component can be the whole pathogen, preferably in an inactivated or attenuated form, an extract of the pathogen or an immunogenic protein of the pathogen.

If the antigen component is an immunogenic protein of the pathogen, that immunogenic protein is preferably expressed in and recovered from in vitro cultured cells.

Therefore, another embodiment relates to a vaccine for preventing or combating infectious disease characterised in that said vaccine comprises an immunostimulating amount of an oligodeoxynucleotide according to the invention and/or a vector according to the invention, an immunogenic amount of an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier.

Of course, the immunostimulating amount of the oligodeoxynucleotide and the immunogenic amount of the antigen component are strongly interrelated. It is one of the merits of the present invention that the presence of the oligodeoxynucleotide according to the invention can lower the amount of antigen component that is necessary to prevent or combat infectious disease. The amount of antigen component that is necessary to prevent or combat infectious disease is referred to as the immunogenic amount of the antigen component.

An immunostimulating amount of the oligodeoxynucleotide is the amount that is capable of decreasing the immunogenic amount of the antigen component, i.e. the amount of the antigen component that is necessary to prevent or combat an infectious disease.

So basically, the wording “immunostimulating amount of the oligodeoxynucleotide” and “immunogenic amount” must be seen in relation to each other.

It goes without saying that, if the vaccine comprises genetic information encoding an antigen component, the amount of antigen component expressed by this genetic information should be enough to prevent or combat infectious disease, i.e.; it must be an immunogenic amount.

The fact that the non-methylated oligodeoxynucleotides according to the invention are immunostimulatory, means that they enhance the immunological efficacy of antigen components in vaccines. For that reason, vaccines according to the invention will in many cases comprise less of the antigen component or the genetic information encoding the antigen component than would be the case if no oligodeoxynucleotides according to the invention would be present.

In some cases an antigen component as such, without the addition of immunostimulatory oligonucleotides, may have such low immunogenic properties that high amounts must be given anyway, albeit without reaching the desired immunogenic level. In such cases, the antigen component can be given in the usual high concentration, however now together with an oligodeoxynucleotide according to the invention in order to so obtain the desired level of immunogenicity.

Thus, the amount of the antigen component or the genetic information encoding the antigen component to be administered with a oligonucleotide according to the invention would as a rule of thumb be equal or below the amount given in the absence of the oligonucleotide. The skilled person involved in the manufacturing of a specific vaccines, would know that amount for that specific vaccine. Also, the Examples give e.g. ample guidance for the amount of antigen components to be used, e.g. in three different inactivated viral vaccines: Newcastle disease virus vaccine, Infectious Bronchitis virus vaccine and Turkey Rhinotracheitis vaccine.

The amount of the oligodeoxynucleotide according to the invention that needs to be administered together with the antigen component or the genetic information encoding the antigen component depends both on the selected oligodeoxynucleotide and the antigen component.

A very suitable amount of oligodeoxynucleotide according to the invention would usually vary between 1 and 100 nanomol. Very good in vivo results have e.g. been obtained with 1-10 μg of oligodeoxynucleotides according to the invention with an average length of 30 deoxynucleotides that were shown to be active in in vitro tests in the nanomolar range.

If an oligodeoxynucleotide is chosen from the group of oligodeoxynucleotides that are active in the picomolar range, the skilled person would realise that amounts below, possibly far below, 1 nanomol, i.e. picomolar amounts, would be worth testing before testing nanomolar amounts.

Vaccines according to the invention comprise a pharmaceutically acceptable carrier. The nature of this carrier depends i.a. upon the route of administration. If the administration route is through the oral or intranasal route, the carrier could be as simple as sterile water, a physiological salt solution or a buffer. If injection is the preferred route, the carrier should preferably be isotonic and have pH restrictions that make it suitable for injection. Such carriers however are extensively known in the art.

Vaccines according to the invention may, in addition to the antigen component or the genetic information encoding the antigen component, and an oligodeoxynucleotide according to the invention, comprise an adjuvant. Adjuvants in general are substances that boost the immune response of the host in a non-specific manner.

Many adjuvants are known in the art to be suitable, such as Freund's Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers and polyanions such as dextran sulphate, carbopol and pyran, alum hydroxide. Also frequently used are alum phosphate, saponins, vegetable oils such as tocopherol and mineral oils. Very efficient adjuvants are oil-in-water emulsions and especially water-in-oil emulsions, further also referred to as are oil-in-water adjuvants and water-in-oil adjuvants. Such emulsions are well-known in the art. Thus, preferably, the vaccine comprises a water-in-oil adjuvant.

Preferably the antigen component is, or is derived from a virus or micro-organism that in its wild-type form is pathogenic to poultry.

More preferably, said virus or micro-organism is selected from the group consisting of Infectious Bronchitis virus, Newcastle Disease virus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent, Avian Reovirus, Mycoplasma gallisepticum, Turkey Rhinotracheitis virus, Haemophilus paragallinarum (Coryza), Chicken Poxvirus, Avian Encephalomyelitis virus, Egg Drop syndrome virus, Infectious Laryngotracheitis virus, Herpes Virus of Turkeys, Eimeria species, Ornithobacterium rhinotracheale, Pasteurella multocida, Mycoplasma synoviae, Salmonella species and Escherichia coli.

Again another embodiment of the present invention relates to an immunostimulatory non-methylated oligodeoxynucleotide according to the invention for use as a medicament

Again another embodiment of the present invention relates to an immunostimulatory non-methylated oligodeoxynucleotide according to the invention for use in preventing or combating infectious disease in poultry

Up till now, all detection systems used transient transfection of cells with the reporter gene. Such transient systems do not allow for a reliable side-by-side comparison of the efficacy of CpG ODN's. As said above, a major improvement over existing systems was the introduction and stable maintenance in cells, of the plasmid carrying the reporter gene. Stable means that the plasmid remains present in the cell after several cell division cycles.

Frequently, stable maintenance of a plasmid is obtained by growing the cells under the pressure of one or more selective agents, such as antibiotics for which a resistance gene is present on the plasmid. Loss of the plasmid would then cause the cell that lost the plasmid to die. Remaining viable cells would still harbour the plasmid.

Another way of stable maintenance would be transfection with linearized plasmids. Such plasmids usually become integrated in the genome of the cell, and thus be stably maintained. Thus, still another embodiment of the present invention relates to a cell comprising a TLR21-receptor and a plasmid encoding an NF-κB reporter gene, which plasmid is stably maintained in the cell. Such cells are very suitable for use in the screening of CpG molecules, more specifically the screening of CpG molecules according to the invention.

The Examples give ample guidance about how to obtain such a cell comprising a plasmid encoding a reporter gene that can be stably maintained in the cell.

As also mentioned above, detection systems based upon secreted alkaline phosphatase (SEAP) were shown to be very suitable for the detection system used.

Thus, preferably the reporter gene is a gene encoding secreted alkaline phosphatase.

Basically, any cell or cell line carrying and expressing a TLR21 that allows introduction and preferably the stable maintenance of a plasmid carrying a NF-κB reporter gene, preferably the SEAP gene as described above is suitable for testing TLR21-specific CpG ODN's.

A preferred example of such a suitable cell line for testing TLR21-specific CpG ODN's is the chicken cell line HD11.

Therefore, preferably, a cell line for use in the detection system is a HD11 cell line comprising a stable plasmid encoding a reporter gene.

Chicken cell lines such as the HD11 cell line display a whole panel of chicken-TLR's. This may in certain conditions generate a certain background activity.

Therefore, non-poultry cell lines such as mammalian cell lines are more preferred cell lines. An example of such a mammalian cell line is a HEK293 cell into which the TLR21 has been cloned. Such a cell line is more specifically selective for TLR21-activating signals.

Therefore, more preferably, a cell line for use in the detection system is the mammalian cell line HEK293 comprising a stably maintained reporter gene and into which HEK293 cell the TLR21 has been cloned.

Still another embodiment of the present invention relates to a method for the detection of immunostimulatory oligodeoxynucleotides according to the invention wherein that method comprises the steps of a) contacting an oligodeoxynucleotide with a cell according to the invention, b) detecting the level of product of the reporter gene.

In a preferred form of this method, the product of the reporter gene is SEAP

A more preferred form of this embodiment relates to a method for the detection of immunostimulatory oligodeoxynucleotides according to the invention, wherein the cell is a cell of chicken cell line HD11, or a HEK293 cell line into which chicken TLR21 has been cloned.

EXAMPLES Example 1 Gene cloning and heterologous expression of chicken TLR21

Recent progress in chicken TLR research suggests that TLR21 is the functional homolog of mammalian TLR9 in avian species (Keestra 2008, Brownlie et al. 2009).

Outline of TLR21 Gene Cloning

Based on the Genbank database sequence NM_(—)001030558, a primer pair was synthesized for the polymerase chain reaction (PCR) amplification of the chicken TLR21 gene:

Ga-TLR21-for1 GAAGCTT ACC ATGATGGAGACAGCGGAGAAGGC Ga-TLR21-rev1 GGCGGCCGC TACATCTGTTTGTCTCCTTCCCTG

The primers were designed to provide flanking restriction cloning sites (underlined) and a Kozak sequence (italic) to the start and stop codons (bold). RT-PCR was performed using these primers and chicken spleen total RNA as a template. A PCR product of the expected size (˜3000 bp) was cloned into pCR2.1-Topo and 5 independent plasmid clones (P1, P2, P12, P13, P14) were sequenced.

DNA sequence of chicken TLR21, as used. AAGCTT ACC ATGATGGAGACAGCGGAGAAGGCATGGCCCAGCACCAGGAT GTGCCCCTCCCACTGCTGTCCACTCTGGCTGCTGCTGCTGGTGACAGTGA CACTGATGCCGATGGTGCACCCGTATGGCTTTCGCAACTGCATTGAGGAT GTCAAGGCACCTTTGTACTTCCGCTGCATCCAGCGCTTCCTGCAGTCGCC GGCCCTGGCAGTGTCTGACCTGCCACCACATGCCATCGCGCTCAATCTGT CATACAACAAAATGCGCTGCCTGCAGCCCTCTGCCTTTGCCCACCTGACA CAGCTGCATACCCTGGACCTGACCTACAACCTCCTGGAGACCCTCTCCCC TGGTGCCTTCAATGGGCTGGGTGTGCTGGTGGTGCTGGACCTGTCTCACA ACAAGCTGACCACACTTGCTGAAGGGGTGTTCAACAGCTTGGGCAACCTG TCCTCGCTGCAGGTACAACATAACCCCCTCAGCACGGTGTCACCAAGTGC TCTGCTACCCCTGGTCAACCTGCGCCGCCTGTCTCTACGGGGCGGGCGGC TGAATGGGTTGGGGGCAGTGGCAGTGGCAGTGCAGGGCTTGGCACAGCTG GAGCTGTTGGACCTATGTGAAAACAACCTGACAACGCTGGGGCCAGGCCC ACCGCTACCCGCCTCGCTGCTCACCCTGCAGCTGTGCAACAACTCGCTGA GGGAGTTAGCGGGGGGCAGCCCGGAGATGCTATGGCACGTGAAGATACTC GACCTCTCCTACAACAGTATCTCACAGGCGGAGGTCTTCACCCAGCTCCA CCTGCGCAACATCAGCCTGCTCCACCTGATCGGCAACCCCTTGGATGTCT TCCACCTGTTGGACATCTCTGACATCCAACCTCGCAGCCTGGATTTCTCT GGGTTGGTGCTGGGGGCTCAGGGGCTGGATAAGGTGTGCCTGAGGCTGCA GGGTCCCCAGGCCTTGCGGCGGCTGCAGCTACAACGCAACGGGCTGAAGG TGCTGCATTGTAATGCACTGCAGTTGTGTCCTGTGCTGAGAGAGCTGGAC CTGTCCTGGAACCGGCTACAGCACGTGGGCTGTGCCGGCCGGCTGCTGGG CAAGAAGCAGCGGGAGAAGCTGGAAGTGCTGACAGTGGAACACAACCTGC TGAAGAAACTGCCGTCTTGCCTGGGGGCCCAGGTGCTGCCTCGGCTGTAC AACATTTCCTTCCGCTTTAACCGCATCCTGACTGTTGGGCCCCAAGCCTT TGCCTACGCCCCGGCCCTGCAGGTGTTGTGGCTCAATATTAACAGCCTGG TGTGGCTGGACAGGCAGGCACTGTGGAGGCTGCACAACCTGACAGAGCTG CGCCTGGACAACAACCTGCTGACCGACCTCTATCACAACTCCTTCATTGA CCTCCACAGACTGCGCACCCTCAACCTGCGCAACAACCGTGTCTCCGTCC TCTTCTCTGGTGTCTTCCAGGGGCTGGCTGAGCTGCAGACGCTGGATTTA GGGGGCAACAACTTGCGCCACCTGACTGCACAGTCACTGCAGGGGCTGCC CAAACTGCGCAGGCTGTACCTGGACCGCAACAGATTGCTGGAGGTGAGCA GCACTGTGTTCGCCCCAGTGCAGGCTACCCTGGGGGTGCTGGACCTGCGG GCCAACAACCTGCAGTACATCTCACAGTGGCTGCGCAAGCCGCCACCCTT CCGCAACCTGAGCAGCCTGTACGACCTGAAGCTGCAGGCGCAGCAGCCCT ATGGACTGAAGATGCTGCCTCACTACTTCTTCCAGGGCTTGGTGAGGCTG CAGCAGCTGTCGCTGTCACAGAACATGCTGCGGTCCATCCCACCGGATGT CTTCGAGGACTTGGGCCAGCTGCGCTCCCTGGCATTGGCTGACAGCAGCA ATGGGCTGCATGACCTGCCTGACGGCATCTTCAGAAACCTGGGCAACCTG CGGTTCCTGGACCTGGAGAATGCAGGGCTGCACTCGCTCACTCTGGAAGT CTTCGGCAATCTCAGCCGGCTGCAGGTGCTGCACTTGGCCAGAAACGAGC TGAAGACCTTCAATGACAGCGTTGCCAGCCGGCTGTCCTCCTTGCGCTAC CTGGACCTGCGCAAGTGTCCGCTCAGCTGCACCTGTGACAACATGTGGCT GCAGGGCTGGCTGAACAACAGCCGTGTGCAGGTTGTCTACCCCTACAACT ACACCTGTGGCTCACAGCACAATGCCTACATCCACAGCTTTGACACACAC GTCTGCTTCCTGGACCTGGGGCTCTATCTCTTTGCTGGGACTGCACCGGC AGTGCTGCTGCTGCTGGTGGTGCCGGTGGTGTACCACCGCGCCTACTGGA GGCTGAAGTACCACTGGTACCTTCTGCGGTGCTGGGTCAACCAGCGGTGG CGGCGGGAGGAAAAGTGCTACCTCTATGACAGCTTTGTGTCCTACAATTC AGCTGATGAAAGTTGGGTGTTGCAGAAGCTGGTGCCTGAGCTGGAGCACG GTGCCTTCCGCCTCTGCTTGCACCACCGCGACTTCCAGCCGGGCCGCAGC ATCATTGACAACATTGTGGATGCTGTCTACAACAGCCGGAAGACGGTGTG CGTGGTGAGCCGCAGCTACCTGCGCAGCGAGTGGTGCTCTCTAGAGGTGC AGTTGGCCAGCTACCGGCTGTTGGATGAGCGGCGTGACATCCTGGTACTG GTGCTGCTGGAGGACGTGGGTGATGCTGAGCTGTCTGCCTACCACCGCAT GCGGCGGGTGCTGCTGCGGCGCACCTACCTGCGCTGGCCTCTTGACCCCG CAGCTCAGCCGCTCTTTTGGGCACGGCTGAAGAGGGCACTGAGGTGGGGA GAGGGAGGAGAGGAGGAGGAAGAAGAAGGTTTGGGTGGAGGGACGGGAAG GCCCAGGGAAGGAGACAAACAGATGTA GCGGCCGC

Transfection of HEK293-pNifTy2-Zeo (clonal cell line) with pcDNA3.1(+)-Neo-chiTLR21

Human embryonic kidney (HEK) cells 293 have been generated in the 1970s by viral transformation (Graham et al., 1977), and are now available to the research community via cell line repositories, such as ATCC.

pNifty2 is a plasmid that allows the detection of NFκB transcription factor activation, which is a hallmark of many immunostimulatory actions, toll-like receptor activations amongst them. The reporter gene in pNifTy2 dependent in its transcription/translation on NFκB activation is secreted alkaline phosphatase (SEAP). Details are described in the datasheet of the company selling this plasmid: Invivogen. Transformation/transfection events by pNifty2 are selected in both bacteria and mammalian cells by zeocin addition to the growth media.

HEK293 cells were transfected with pNifTy2 by standard methods (lipofection), a stable cell line was selected, the functionality of the NF-κB/SEAP axis established by stimulation with human tumor necrosis factor α (Sigma). Secreted SEAP in the culture supernatant of stimulated cells was determined by a microtiter plate colorimetric assay employing the chromogenic substrate p-nitrophenylphosphate (pNPP, 5 mM) in an alkaline buffer (50 mM NaHCO₃, pH9.6, 2 mM MgCl₂). Colour development (λ=405 nm) was monitored by a microtiter plate reader. This readout was also used for selecting clonal lines (by the limiting dilution method) with high signal to noise ratios. One of these selected clones (dubbed clone 11) was then used for further studies with chicken TLR21.

pcDNA3.1(+)-neo is a standard mammalian expression vector purchased from Invitrogen. Subcloning of the chicken TLR21 gene into this vector was done via flanking Hind III (start codon) and Not I (stop codon) sites that were introduced by PCR. (See FIG. 1).

This plasmid was then transfected (lipofection) into the clonal HEK293-pNifty2-zeo line, and recombinant cells were selected by addition of both zeocin and G418 into the growth medium. Functionality of the resulting polyclonal recombinant cell line was assessed by stimulation of the culture with ODN-X4 and ODN-HEK1-PTO and detection of SEAP. Superior clonal lines were then identified by the limiting dilution method followed by stimulation and SEAP detection.

SEAP is a reporter enzyme in mammalian systems (Yang et al., 1997). SEAP is a secreted form of human embryonic alkaline phosphatase. Its main advantages are the high stability and the extremely high specific activity, which ensure sensitivity and robustness of detection. Several substrates have been described for SEAP detection, but the economical and robust pNPP was selected, as its reaction product p-nitrophenolate is detected with high sensitivity (ε₄₀₅=18500 M⁻¹ cm⁻¹). In our test setups, we perform kinetic assays, because they provide a wider dynamic range of quantification.

HEK293-pNifTy2-Zeo cells were transfected with pcDNA3.1(+)-Neo-chiTLR21 (linearized with Pvu I) and a polyclonal cell line was selected by supplementing the media with 350 μg/ml zeocin and 600 μg/ml G418. A functionality test was performed by stimulating the cells with ODN-X4 (PDE) and with ODN-HEK1 (PTO). Secreted alkaline phosphatase (SEAP) was produced by the selected cells, but not by the parental HEK293-pNifTy2-Zeo cell line. Single cell cloning was performed and individual clones were analyzed for their responsiveness to ODN-X4 (PDE) (GGGGGGTTCGTTTTCGTTTTCGTTGGGGG) and ODN-HEK1 (PTO) (TCGTCGTTTTGTCGTTTGTCGTT).

Out of 46 zeo/G418-double-resistant clonal cell lines, only 3 were clearly responsive to the ODN stimuli, while 3-4 further cell lines showed weaker signals. 85% of the selected clones were, therefore, not functional.

For all further studies, clonal cell line 38, which produced by far the highest SEAP readout signal on response to ODN-X4 (PDE) and ODN-HEK1 (PTO) stimulation, was used.

FIGS. 2-5 give an overview of the SEAP activity of the various zeo/G418-double-resistant clonal cell lines.

Example 2

Starting from X43-batch4: GGGGGGTTCGTCTTCGTCTTCGTCGGGGG, the following modifications were made and tested:

X43-I-T

X43-I-C

X43-II- T

X43-II- C

X23- GGGGGGGTCGTCGTCGTCGTCGTCGGGGG batch3 X23-I

As can be seen from FIG. 6, oligonucleotide X43-II-T gives an extreme induction of the amount of the colored product absorbing at 405 nm of the reporter enzyme SEAP in the reporter cells (HEK293-pNifty2-chickenTLR21) with regard to maximal response, when compared to the other oligonucleotides tested.

X43-II-T represents the formula ^(5′) [G]_(x) {[T]_(p) T T C G T C [T]_(q)}_(n)[G]_(z) ^(3′) wherein x=6, z=5, p=2, q=2 and n=3.

On the basis of FIG. 6, the following EC₅₀ calculation were made for the various compounds:

EC₅₀ calculation: X43-batch4 3.91 nM X43-I-T >1200 nM X4-I-C >300 nM X43-II-T 0.53 nM X43-II-C 8.28 nM X23-batch3 6.08 nM X23-I >400 nM

Conclusion: as follows from FIG. 6 representing the OD405nm/min and FIG. 7 representing the EC₅₀ in nanomolar, the addition of two 3′-flanking thymidines and two 5′-flanking thymidines in X43 (→X43-II-T) leads to an unexpectedly strong increase of potency both with respect to maximal response and to EC₅₀. The EC₅₀ of the compound ^(5′) [G]₆ {[T]₂ T T C G T C [T]₂}₃ [G]₅ ^(3′) was shown to be in the picomolar range.

Example 3

In this Example, the number of n was varied from n=3 in X43-II-trip, to n=4 (X43-II-quad) and to n=5 (X43-II-pent)

X43-trip 5′-GGGGGGTTCGTCTTCGTCTTCGTCGGGGG-3′+0(= standard 1) X4-pent 5′-GGGGGGTTCGTTTTCGTTTTCGTTTTCGTTTTCGTTGGGGG-3′ (= standard 2) X43-II-trip 5′-GGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTGGGGG-3′ (n = 3, and with [p and q] = 2) X43-II-quad 5′-GGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTGGGGG-3′ n = 4, and with [p and q] = 2) X43-II-pent 5′-GGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTGGGGG-3′ n = 5, and with [p and q] = 2)

In FIG. 8, the effect of the increase in the number of n is shown. In the table below, the effect of an increase in n on the EC₅₀ is shown. It is clear that an increase in n leads to a decrease in EC₅₀ and thus to a higher immunostimulatory effect.

ODN EC₅₀ X43-trip 338 pM X43-II-trip 155 pM X43-II-quad 132 pM X43-II-pent  59 pM X4-pent 340 pM

Example 4

In this Example, the number of p and q was changed from 2 (X43-II-trip) to 3 (X43-III-trip) and to 4 (X43-IV-trip)

X43-I-trip 5′-GGGGGGTTCGTCTTCGTCTTCGTCGGGGG-3′ (= standard 1) (n = 3, and with [p and q] = 0) X4-pent 5′-GGGGGGTTCGTTTTCGTTTTCGTTTTCGTTTTCGTTGGGGG-3′ (= standard 2) X43-II-trip 5′-GGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTTGGGGG-3′ (n = 3, and with [p and q] = 2) X43-III-trip 5′-GGGGGGTTTTTCGTCTTTTTTTTCGTCTTTTTTTTCGTCTTTGGGGG-3′ (n = 3, and with [p and q] = 3) X43-IV-trip 5′-GGGGGGTTTTTTCGTCTTTTTTTTTTCGTCTTTTTTTTTTCGTCTTTTGGGGG-3′ (n = 3, and with [p and q] = 4)

In FIG. 9, the effect of the increase in the number of p and q is shown. In the table below, the effect of an increase in n on the EC₅₀ is shown. It is clear that an increase in p and q leads to a decrease in EC₅₀ and thus to a higher immunostimulatory effect.

ODN EC₅₀ X43-trip 338 pM X43-II-trip 155 pM X43-III-trip 148 pM X23-IV-trip 104 pM X4-pent 340 pM

Example 5

In this Example, the number of x was increased from 6 to 7 (X43-II-5735) and z was decreased from 5 to 2 (X43-II-5732).

X43-II 5′-GGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTT GGGGG-3′ (= standard 1, X43-II-trip) X4-pent 5′-GGGGGGTTCGTTTTCGTTTTCGTTTTCGTTTTCGTT GGGGG-3′ (= standard 2) X43-II-5735 5′-GGGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTT GGGGG-3′ (→ x = 7, z = 5, p and q both = 2 and n = 3) X43-II-5732 5′-GGGGGGGTTTTCGTCTTTTTTCGTCTTTTTTCGTCTT GG-3′ (→ x = 7, z = 2, p and q both = 2 and n = 3)

In FIG. 10, the effect of the increase in the number of x and the decrease in the number of z is shown. In the table below, the effect of an increase in n on the EC₅₀ is shown. An increase of x and a decrease of z both lead to an increase in EC50 and thus to a higher immunostimulatory effect.

ODN EC₅₀ X43-II-trip 155 pM X43-II-5735 105 pM X23-II-5732 <<100 pM   X4-pent 340 pM

Legend to the Figures

FIG. 1: Plasmid map of pcDNA3.1(+)-chiTLR21

FIG. 2-5: overview of the SEAP activity of the various zeo/G418-double-resistant clonal cell lines.

FIG. 6: this figure shows the strength of the various compounds tested, in inducing an amount of the colored product absorbing at 405 nm of the reporter enzyme SEAP in the reporter cells (HEK293-pNifty2-chickenTLR21)

FIG. 7: this figure shows the EC₅₀ values of the various compounds tested, in picomoles.

FIGS. 8-10: these figures show the strength of further modified compounds tested, in inducing an amount of the colored product absorbing at 405 nm of the reporter enzyme SEAP in the reporter cells (HEK293-pNifty2-chickenTLR21).

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1. An immunostimulatory non-methylated oligodeoxynucleotide having the general formula ^(5′) [G]_(x) {[T]_(p) T T C G T C [T]_(q)}_(n) [G]_(z) ^(3′) wherein p=1-15, q=1-15, n=2-100, x=3-10 and z=0-10 or a pharmaceutically acceptable salt thereof.
 2. The oligodeoxynucleotide according to claim 1, wherein p>1.
 3. The oligodeoxynucleotide according to claim 1, wherein p>2.
 4. The oligodeoxynucleotide according to claim 1, wherein q>1.
 5. The oligodeoxynucleotide according to claim 1, wherein q>2.
 6. The oligodeoxynucleotide according to claim 1, wherein n=3-18
 7. The oligodeoxynucleotide according to claim 1, characterised in that the ^(5′) [G]_(x) and the ^(3′) [G]_(z) nucleotides have a phosphorothioate binding and the other nucleotides have a phosphodiester binding.
 8. The oligodeoxynucleotide according to claim 1, wherein {[T]_(p) T T C G T C [T]_(q)}_(n) is a homopolymer.
 9. The oligodeoxynucleotide according to claim 1, wherein said oligodeoxynucleotide is coupled to a carrier or hapten.
 10. A vector comprising the oligodeoxynucleotide according to claim
 1. 11. A vaccine for preventing or combating an infectious disease, characterised in that said vaccine comprises an immunostimulatory amount of an oligodeoxynucleotide according to claim 1, an immunological amount of an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier.
 12. The vaccine according to claim 11, characterised in that said antigen component is, or is derived from a virus or micro-organism that in its wild-type form is pathogenic to poultry.
 13. The vaccine, according to claim 12 characterised in that-said virus or micro-organism is selected from the group consisting of Infectious Bronchitis virus, Newcastle Disease virus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent, Avian Reovirus, Mycoplasma gallisepticum, Turkey Rhinotracheitis virus, Haemophilus paragallinarum (Coryza), Chicken Poxvirus, Avian Encephalomyelitis virus, Egg Drop syndrome virus, Infectious Laryngotracheitis virus, Herpesvirus of Turkeys, Eimeria species, Omithobacterium rhinotracheale, Pasteurella multocida, Mycoplasma synoviae, Salmonella species and E. coli.
 14. An immunostimulatory non-methylated oligodeoxynucleotide according to claim 1 for use as a medicament.
 15. An immunostimulatory non-methylated oligodeoxynucleotide according to claim 1 for use in the prevention of infection in poultry.
 16. A method for the detection of immunostimulatory oligodeoxynucleotides according to claim 1, characterised in that said method comprises the steps of a) contacting an oligodeoxynucleotide with a cell comprising a TLR21-receptor and a plasmid encoding an NF-κB reporter gene, which plasmid is stably maintained in the cell, and b) detecting the level of the product of the reporter gene.
 17. A method for the detection of immunostimulatory oligodeoxynucleotides according to claim 1, characterised in that said method comprises the steps of a) contacting an oligodeoxvnucleotide with a cell comprising a TLR21-receptor and a plasmid encoding an NF-κB reporter gene, which plasmid is stably maintained in the cell, and b) detecting the level of the product of the reporter gene, wherein the product of the reporter gene is secreted alkaline phosphatase.
 18. A method for the detection of immunostimulatory oligodeoxynucleotides according to claim 1, characterised in that said method comprises the steps of a) contacting an oligodeoxynucleotide with a cell comprising a TLR21-receptor and a plasmid encoding an NF-KB reporter gene, which plasmid is stably maintained in the cell, and b) detecting the level of the product of the reporter gene, wherein the cell is a cell of chicken cell line HD11, or a HEK293 cell line into which a chicken TLR21 receptor gene has been cloned.
 19. A vaccine for preventing or combating an infectious disease, characterised in that said vaccine comprises vector comprising the oligodeoxynucleotide according to claim 1, an immunological amount of an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier.
 20. A vaccine for preventing or combating an infectious disease, characterised in that said vaccine comprises vector comprising the oligodeoxynucleotide according to claim 1, an immunological amount of an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier, wherein said antigen component is, or is derived from a virus or micro-organism that in its wild-type form is pathogenic to poultry.
 21. A vaccine for preventing or combating an infectious disease, characterised in that said vaccine comprises vector comprising the oligodeoxynucleotide according to claim 1, an immunological amount of an antigen component or genetic information encoding an antigen component, and a pharmaceutically acceptable carrier, wherein said antigen component is, or is derived from a virus or micro-organism that in its wild-type form is pathogenic to poultry, and wherein said virus or micro-organism is selected from the group consisting of Infectious Bronchitis virus, Newcastle Disease virus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent, Avian Reovirus, Mycoplasma gallisepticum, Turkey Rhinotracheitis virus, Haemophilus paragallinarum (Coryza), Chicken Poxvirus, Avian Encephalomyelitis virus, Egg Drop syndrome virus, Infectious Laryngotracheitis virus, Herpesvirus of Turkeys, Eimeria species, Omithobacterium rhinotracheale, Pasteurella multocida, Mycoplasma synoviae, Salmonella species and E. coli.
 22. A vector comprising the oligodeoxynucleotide according to claim 1 for use as a medicament.
 23. A vector comprising the oligodeoxynucleotide according to claim 1 for use in the prevention of infection in poultry. 